Drosophila mushroom body (MB) γ neurons undergo axon pruning during metamorphosis through a process of localized degeneration of specific axon branches. Developmental axon degeneration is initiated by the steroid hormone ecdysone, acting through a nuclear receptor complex composed of Ultraspiracle (USP) and Ecdysone Receptor B1 (EcRB1) to regulate gene expression in MB γ neurons. To identify ecdysone-dependent gene expression changes in MB γ neurons at the onset of axon pruning, we use laser-capture microdissection to isolate wild-type and mutant MB neurons in which EcR activity is genetically blocked, and analyze expression changes by microarray. We identify several molecular pathways that are regulated in MB neurons by ecdysone. The most striking observation is the upregulation of genes involved in the ubiquitin-proteasome system (UPS), which is cell-autonomously required for γ neuron pruning. In addition, we characterize the function of Boule, an evolutionarily conserved RNA-binding protein previously implicated in spermatogenesis in flies and vertebrates. boule expression is downregulated by ecdysone in MB neurons at the onset of pruning, and forced expression of Boule in MB γ neurons is sufficient to inhibit axon pruning. This activity is dependent on Boule’s RNA-binding domain and a conserved DAZ domain implicated in interactions with other RNA-binding proteins. However, loss of Boule does not result in obvious defects in axon pruning or morphogenesis of MB neurons, suggesting that it acts redundantly with other ecdyonse-regulated genes. We propose a novel function for Boule in the central nervous system as a negative regulator of developmental axon pruning.
Keywords: axon degeneration, mushroom body, neural development, ecdysone, ecdysone receptor, Ubiquitin Proteasome System, metamorphosis